Human serum albumin (HSA) is a globular, non-glycosylated protein (MW 65,000) synthesized by the liver. Circulating in the blood stream at levels of 42 gr/liter, it is the most abundant serum protein. HSA is involved in a number of essential functions, including sustaining normal bloodstream osmolarity, regulating blood pressure and transporting fatty acids, amino acids, bile pigments and numerous small molecules. Clinically, HSA is used in large quantities to replace blood volume in acute phase conditions such as trauma and severe burns or surgical procedures. Currently, medical supply of HSA depends on the fractionation of donated human blood. At the present time, the cost of purifying HSA from blood is relatively low, since HSA as well as other blood products can be simultaneously purified from the same source. However, as other blood products, such as coagulation factors, are produced by biotechnology instead of purified from human blood, market dynamics will increase the relative cost of purification of HSA from blood. The threat of a diminishing supply of donated blood, rising costs of purifying HSA from blood and the potential risk of contamination with infectious viruses that cause hepatitis, AIDS and other diseases make an alternative source of production of large quantities of HSA desirable. As such, alternative approaches to the production of large quantities of HSA are required.
Recombinant DNA technology has been used increasingly over the past decade for the production of commercially important biological materials. To this end, the DNA sequences encoding a variety of medically important human proteins have been cloned. These include insulin, plasminogen activator, alpha-antitrypsin and coagulation factors VIII and IX.
The expression of DNA sequences encoding these and other proteins has been suggested as the ideal source for the production of large quantities of mammalian proteins. A variety of hosts have been utilized for the production of medically important proteins including bacteria yeast, cultured cells and animals. In practice, bacteria and yeast often prove unsatisfactory as hosts because the foreign proteins are often unstable and are not processed correctly. However, in bacteria the HSA is produced as an insoluble aggregate which requires processing to yield the mature, soluble protein. HSA has also been produced in yeast but at significantly lower levels and in which a high proportion is either fragmented, cell associated or insoluble (Sleep et al, 1990, Bio/technology 8:42-46; Etcheverry et al., 1986, Bio/Technology 4:729-730; Quirk et al., 1989, Biotech. and Applied Biochem. 11:273-287).
In light of this problem, the expression of cloned genes in mammalian tissue culture has been attempted and has, in some instances, proved a viable strategy. However, batch fermentation of animal cells is an expensive and technically demanding process. Transgenic animals have also been proposed as a source for the production of protein products. The production of transgenic livestock offers a number of potential applications including "Molecular Farming" (also referred to as Genetic Farming) where proteins of medical or commercial importance are targeted for high level expression and production in the mammary gland with subsequent secretion into the milk of such genetically engineered animals. The feasibility of this approach was first tested in transgenic mice.
WO-A-8800239 discloses transgenic animals which secrete a valuable pharmaceutical protein, in this case Factor IX, into the milk of transgenic sheep. EP-A-0264166 also discloses the general idea of transgenic animals secreting pharmaceutical proteins into their milk.
Early work with transgenic animals, as represented by WO-A-8800239 has used genetic constructs based on cDNA coding for the protein of interest. The cDNA will be smaller than the natural gene, assuming that the natural gene has introns, and for that reason is easier to manipulate. It is desirable for commercial purposes to improve upon the yields of proteins produced in the milk of the transgenic animal.
Brinster et al (PNAS 85 836-840 (1988)) have demonstrated that the transcriptional efficiency of transgenes having introns in transgenic mice is increased over that of cDNA. Brinster et al show that all the exons and introns of a natural gene are important both for efficient and for reliable expression (that is to say, both the levels of the expression and the proportion of expressing animals) and is due to the presence of the natural introns in that gene. It is known that in some cases this is not attributable to the presence of tissue-specific regulatory sequences in introns, because the phenomenon is observed when the expression of a gene is redirected by a heterologous promoter to a tissue in which it is not normally expressed. Brinster et al say that the effect is peculiar to transgenic animals and is not seen in cell lines. However, Huang and Gorman (1990, Nucleic Acids Research 18:937-947) have demonstrated that a heterologous intron linked to a reporter gene can increase the level of expression of that gene in tissue culture cells.
The problems of yield and reliability of expression can not be overcome by merely following the teaching of Brinster et at and inserting into mammalian genomes transgenes based on natural foreign genes as opposed to foreign cDNA. First, as mentioned above, natural genes having introns are larger than the cDNA coding for the product of the gene since the introns are removed from the primary transcription product before export from the nucleus as mRNA. It is technically difficult to handle large genomic DNA.
Secondly, the longer the length of manipulated DNA, the greater chance that restriction sites occur more than once, thereby making manipulation more difficult. This is especially so given the fact that in most transgenic techniques, the DNA to be inserted into the mammalian genome will often be isolated from prokaryotic vector sequences (because the DNA will have been manipulated in a prokaryotic vector, for choice). The prokaryotic vector sequences usually have to be removed, because they tend to inhibit expression. So the longer the piece of DNA, the more difficult it is to find a restriction enzyme which will not cleave it internally.
Attempts to achieve protein expression utilizing cDNA encoding the protein instead of the full length gene, have generally resulted in low protein yields. A number of workers recognized the desirability of improving upon the yields and reliability of transgenic techniques obtained when using constructs based on cDNA.
Archibald et al. (WO90/05188) noted that with certain proteins higher yields (than could be obtained utilizing cDNA) could be obtained when at least some of the naturally occurring introns were utilized. Palmiter et al. (1991, Proc. Natl. Acad. Sci, USA 88:478-482) also found that the level of expression of a transgene was higher when the transgene included some introns as compared with the transgene composed of a cDNA. However, the level of expression with less then all of its natural introns was reduced when compared to the level of expression obtained with the entire gene with all of its introns.